Extensive research has been conducted on the isolation of DNA polymerases from mesophilic microorganisms such as E. coli. See, for example, Bessman et al., 1957, J. Biol. Chem. 223:171-177, and Buttin and Kornberg, 1966, J. Biol. Chem. 241:5419-5427.
Interest in DNA polymerases from thermophilic microbes increased with the invention of nucleic acid amplification processes. The use of thermostable enzymes, such as those described in U.S. Pat. No. 4,165,188, to amplify existing nucleic acid sequences in amounts that are large compared to the amount initially present was described U.S. Pat. Nos. 4,683,195 and 4,683,202, which describe the PCR process. These patents are incorporated herein by reference. The PCR process involves denaturation of a target nucleic acid, hybridization of primers, and synthesis of complementary strands catalyzed by a DNA polymerase. The extension product of each primer becomes a template for the production of the desired nucleic acid sequence. These patents disclose that, if the polymerase employed is a thermostable enzyme, then polymerase need not be added after every denaturation step, because heat will not destroy the polymerase activity.
The thermostable DNA polymerase from Thermus aquaticus (Taq) has been cloned, expressed, and purified from recombinant cells as described in Lawyer et al., 1989, J. Biol. Chem. 264:6427-6437, and U.S. Pat. Nos. 4,889,818 and 5,079,352, which are incorporated herein by reference. Crude preparations of a DNA polymerase activity isolated from T. aquaticus have been described by others (Chien et al., 1976, J. Bacteriol. 127:1550-1557, and Kaledin et al., 1980, Biokymiya 45:644-651).
U.S. Pat. No. 4,889,818, European Patent Publication No. 258,017, and PCT Publication No. 89/06691, the disclosures of which are incorporated herein by reference, all describe the isolation and recombinant expression of an .about.94 kDa thermostable DNA polymerase from Thermos aquaticus and the use of that polymerase in PCR. Although T. aquaticus DNA polymerase is especially preferred for use in PCR and other recombinant DNA techniques, a number of other thermophilic DNA polymerases have been purified, cloned, and expressed. (See co-pending, commonly assigned PCT Patent Publication Nos. WO 91/09950, WO 92/03556, WO 92/06200, and WO 92/06202, which are incorporated heroin by reference.)
Thermostable DNA polymerases are not irreversibly inactivated even when heated to 93.degree.-95.degree. C. for brief periods of time, as, for example, in the practice of DNA amplification by PCR. In contrast, at this elevated temperature E. coli DNA Pol I is inactivated.
Archaeal hyperthermophiles, such as Pyrodictium and Methanopyrus species, grow at temperatures up to about 110.degree. C. and are unable to grow below 80.degree. C. (see, Stetter et al., 1990, FEMS Microbiology Reviews 75:1170124, which is incorporated herein by reference). These sulfur reducing, strict anaerobes are isolated from submarine environments. For example, P. abyssi was isolated from a deep sea active "smoker" chimney off Guaymas Mexico at 2,000 meters depth and in 320.degree. C. of venting water (Pley et al., 1991, Systematic and Applied Microbiology 14:245). In contrast to the Pyrodictium species, other thermophilic microorganisms having an optimum growth temperature at or about 90.degree. C. and a maximum growth temperature at or about 100.degree. C. are not difficult to culture. For example, a gene encoding DNA polymerase has been cloned and sequenced from Thermococcus litoralis (EP No. 455,430).
In contrast, culture of the extreme hyperthermophilic microorganisms is made difficult by their inability to grow on agar solidified media. Individual cells of the Pyrodictium species are extremely fragile, and the organisms grow as fibrous networks. Standard bacterial fermentation techniques are extremely difficult for culturing Pyrodictium species due to the fragility of the cells and tendency of the cells to grow as networks clogging the steel parts of conventional fermentation apparatus. (See Staley, J. T. et al. eds., Bergey's Manual of Systematic Bacteriology, 1989, Williams and Wilkins, Baltimore, which is incorporated herein by reference.) These difficulties preclude laboratory culture for preparing large amounts of purified nucleic acid polymerase enzymes for characterization and amino acid sequence analysis. Those skilled in the art may be able to culture Pyrodictium to a cell density approaching 10.sup.6 -10.sup.7 cells/ml (see, for example, Phipps et al., 1991, EMBO J. 10(7):1711-1722). In contrast, E. coli is routinely grown to 0.3-1.0.times.10.sup.11 cells/ml.
Accordingly, there is a need for the characterization, amino acid sequence, DNA sequence, and expression in a non-native host, of hyperthermophile DNA polymerase enzymes to eliminate the prior difficulties associated with the native host. In addition there is a desire in the art to produce thermostable DNA polymerases having enhanced thermostability that may be used to improve the PCR process and to improve the results obtained when using a thermostable DNA polymerase in other recombinant techniques such as DNA sequencing, nick-translation, and reverse transcription.
The present invention meets these needs by providing DNA and amino acid sequence information, recombinant expression vectors and purification protocols for DNA polymerases from Pyrodictium species.